Galidesivir Triphosphate Promotes Stalling of Dengue-2 Virus Polymerase Immediately Prior to Incorporation

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 μM and 47 ± 5 μM, respectively, at an ATP concentration of 20 μM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.


Section A: Supplementary Figures
Supplementary Figure S3: Comparison of the RdRp activity of DENV2 NS5 in the presence of Mn 2+ and Mg 2+ ions.Reactions contained 200 nM DENV2 NS5, 2.5 µM SYTO 9, 40 µg/ml poly(U), 0 to 10 mM MnCl2 or MgCl2 and were initiated with 0.5 mM ATP. Measured rates in fluorescence units per second are given as a percentage relative to the maximum rate for that experiment.Technical triplicates are shown, connected by a line passing through the median of each set of triplicates.

Mg 2+
Supplementary Figure S4: Optimization of divalent metal ion concentration for the primer extension assay.Primer extension assay was performed as described in methods using template B (Figure 8A) and in presence of only 10 µM GTP.Lane 1 is the primer by itself.Lane 2-7 are reactions performed using different concentrations of MgCl2 and MnCl2 as indicated.The divalent metal ion concentrations selected for the primer extension assay is highlighted by a green asterisk.
Supplementary Figure S5: Time course of a primer extension reaction with template G (Table 4), analyzed using denaturing polyacrylamide gel electrophoresis, together with band intensity as a function of fluorescently-labeled primer concentration.The leftmost lanes show the band intensity resulting from control reactions lacking NTPs, while incorporating the fluorescently-labeled primer at the indicated concentrations.The rightmost lanes show the product distribution obtained using template G at the indicated times following initiation of the reaction with 5 µM each of ATP, GTP, CTP and UTP.
Supplementary Figure S6: Unedited gel image of the results of the primer extension assay analyzed by denaturing polyacrylamide gel electrophoresis.Lane 1 is the primer by itself.Control reactions (lanes 2-6) were performed using template G. Reactions to investigate the effects of Galidesivir incorporation (lanes 7-20) were performed using templates A-F.Full details are given in Figure 8 of the article.
Supplementary Figure S7: Unedited gel image of the results of the primer extension assay using templates H-K analyzed by denaturing polyacrylamide gel electrophoresis.Full details are given in Figure 9   ).The poly(A):poly(U) concentration was varied from 0 to 8 µg/ml.This corresponds to an effective A:U base pair concentration of 0 to 12.6 x 10 -6 M, given a molecular weight of 633.07 gmol -1 for each A:U base pair unit.
of the article.Supplementary Figure S8.Schematic diagram showing the detail of NS5 gene insertion into the multiple cloning site of vector pET15b(+) (Novagen).
Ser His His His His His His Thr Ser Gly Ser Gly Ser Glu Asn Leu Tyr Phe Gln Gly Thr Gly … Val Leu Trp ---ACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC ACT AGT GGG AGT GGC AGC GAA AAC CTG TAT TTT CAG GGA ACT GGC … GTT CTG TGG TGA CTC GAG Ser His His His His His His Thr Ser Gly Ser Gly Ser Glu Asn Leu Tyr Phe Gln Gly Gly Gly … Gly Val Leu ---ACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC ACT AGT GGG AGT GGC AGC GAA AAC CTG TAT TTT CAG GGA GGT GGG … GGA GTG TTG TGA CTC GAGS6Supplementary FigureS9: Example of a poly(A):poly(U) standard curve in the NS5 RdRp assay conditions (200 nM DENV2 NS5, 20 µM A2 (ApA) primer, 40 µg/ml poly(U), 2.5 mM MnCl2, 3 µM SYTO 9 Reaction progress was monitored by thin layer chromatography (TLC) on Merck Aluminum-backed silica gel coated TLC plates (60 Å, F254 indicator).TLC plates were visualized by exposure to ultraviolet light (254 nm), and/or staining with ceric ammonium molybdate stain (Hanessian's Stain) or KMnO4 stain.Flash column chromatography was performed with a Büchi Pure C-815 Flash automated flash chromatography system using prepacked FlashPure cartridges containing either silica gel (50 μm irregular) or C18 silica gel (50 μm spherical), and ACS grade solvents.NMR spectra were recorded using a Bruker 500 MHz spectrometer and analyzed using MestReNova software.Data are represented as follows: chemical shift (δ) in parts per million (ppm), multiplicity (s = singlet, d = doublet, dd (doublet of doublets), t = triplet, q = quartet, m = multiplet), coupling constants (J) in Hertz(Hz), and integration.High resolution electrospray ionization (ESI) mass spectrometric analysis and liquid chromatographymass spectrometric analysis (LC-MS) were performed on Waters Q-TOF Premier™ Tandem Mass spectrometer fitted with a Waters 2795 HPLC and analyzed using MassLynx software.LCMS was performed using a ACQUITY UPLC™ BEH C18 column (1.7 µm, 100 × 2.1 mm, 130 Å) and method that used: mobile phase (A: 10 mM ammonium formate in H2O; B: MeOH), flow rate (0.2 mL/min), temperature (30 °C), and detection method (diode array).